Abstract Rhabdomyosarcoma (RMS), a cancer related to the skeletal muscle lineage, is the most common soft-tissue sarcoma of childhood and adolescence. Despite intergroup clinical trials, survival for high-risk groups has not improved in over four decades. Children with the alveolar variant of RMS, and accompanied by the fusion protein PAX3-FOXO1 (PF), have a particularly dismal outcome of <10% survival when metastatic. An improved understanding of the aberrant signaling underlying PF-RMS is vital for the development of new therapies. This work seeks to exploit a novel genetically engineered mouse model (GEMM) of PF-RMS that was recently developed in our laboratory. This new 9-allele GEMM, in which both isoforms of the Hippo kinase (also known as MST tumor suppressor) are deleted, generates PF-RMS tumors with a shorter latency and increased aggressiveness compared to earlier GEMMs. This project evolved from our prior investigations showing that PF contributes to RMS in part by disabling the Hippo tumor suppressor pathway. In summary, we defined a PF?RASSF4 ?MST axis, in which PF transcriptionally upregulates expression of a scaffold protein known as RASSF4, which in turn binds to and inhibits the Hippo tumor suppressor kinase MST1. To build upon these findings, we sought to evaluate the consequences of conditional loss of both MST1 and MST2 alleles in a GEMM of PF-RMS. The resulting GEMM (which we term MSTNull) is the product of this effort. Important observations from this new GEMM include that loss of MST accelerates PF-RMS tumorigenesis and increases penetrance, and signals not to the canonical Hippo protein YAP1, but to the little-studied protein MOB1. We do not know how MST loss contributes to PF-RMS tumorigenesis. Nor do we know whether silencing of MST1 or MST2 is more critical. Finally, although we have zeroed in on non-canonical signaling, we do not know which genes/proteins are downstream from this PF?RASSF4 ?MST axis. We therefore propose the following Specific Aims: (1) determine the cellular phenotypic consequences of MST loss in PF-RMS, (2) investigate the unique contributions of MST1 and MST2 loss to PF-RMS, and (3) identify the downstream genes/proteins that mediate the increased tumorigenesis of the PF?RASSF4 ?MST axis. Completion of these aims will provide critical foundational information on the mechanisms through which the PF?RASSF4 ?MSTaxis supports PF- RMS tumorigenesis. The fundamental goal is to identify novel therapeutic targets for this difficult-to-cure sarcoma.